The present invention provides recombinant methods and materials for producing novel polyketides by recombinant DNA technology. The invention relates to the fields of agriculture, animal husbandry, chemistry, medicinal chemistry, medicine, molecular biology, pharmacology, and veterinary technology.
Polyketides represent a large family of diverse compounds synthesized from 2-carbon units through a series of condensations and subsequent modifications. Polyketides occur in many types of organisms, including fungi and mycelial bacteria, in particular, the actinomycetes. There are a wide variety of polyketide structures, and the class of polyketides encompasses numerous compounds with diverse activities. Erythromycin, FK-506, FK-520, megalomicin, narbomycin, oleandomycin, picromycin, rapamycin, spinocyn, and tylosin are examples of such compounds. Given the difficulty in producing polyketide compounds by traditional chemical methodology, and the typically low production of polyketides in wild-type cells, there has been considerable interest in finding improved or alternate means to produce polyketide compounds. See PCT publication Nos. WO 93/13663; WO 95/08548; WO 96/40968; 97/02358; and 98/27203; U.S. Pat. Nos. 4,874,748; 5,063,155; 5,098,837; 5,149,639; 5,672,491; and 5,712,146; Fu et al., 1994, Biochemistry 33: 9321-9326; McDaniel et al., 1993, Science 262: 1546-1550; and Rohr, 1995, Angew. Chem. Int. Ed. Engl. 34(8): 881-888, each of which is incorporated herein by reference.
Polyketides are synthesized in nature by polyketide synthase (PKS) enzymes. These enzymes, which are complexes of multiple large proteins, are similar to the synthases that catalyze condensation of 2-carbon units in the biosynthesis of fatty acids. PKS enzymes are encoded by PKS genes that usually consist of three or more open reading frames (ORFs). Two major types of PKS enzymes are known; these differ in their composition and mode of synthesis. These two major types of PKS enzymes are commonly referred to as Type I or xe2x80x9cmodularxe2x80x9d and Type II xe2x80x9citerativexe2x80x9d PKS enzymes.
Modular PKSs are responsible for producing a large number of 12-, 14-, and 16-membered macrolide antibiotics including erythromycin, megalomicin, methymycin, narbomycin, oleandomycin, picromycin, and tylosin. Each ORF of a modular PKS can comprise one, two, or more xe2x80x9cmodulesxe2x80x9d of ketosynthase activity, each module of which consists of at least two (if a loading module) and more typically three (for the simplest extender module) or more enzymatic activities or xe2x80x9cdomains.xe2x80x9d These large multifunctional enzymes ( greater than 300,000 kDa) catalyze the biosynthesis of polyketide macrolactones through multistep pathways involving decarboxylative condensations between acyl thioesters followed by cycles of varying xcex2-carbon processing activities (see O""Hagan, D. The polyketide metabolites; E. Horwood: New York, 1991, incorporated herein by reference).
During the past half decade, the study of modular PKS function and specificity has been greatly facilitated by the plasmind-based Streptomyces coelicolor expression system developed with the 6-deoxyerythronolide B (6-dEB) synthase (DEBS) genes (see Kao et al., 1994, Science, 265: 509-512, McDaniel et al., 1993, Science 262: 1546-1557, and U.S. Pat. Nos. 5,672,491 and 5,712,146, each of which is incorporated herein by reference). The advantages to this plasmid-based genetic system for DEBS are that it overcomes the tedious and limited techniques for manipulating the natural DEBS host organism, Saccharopolyspora erythraea, allows more facile construction of recombinant PKSs, and reduces the complexity of PKS analysis by providing a xe2x80x9ccleanxe2x80x9d host background. This system also expedited construction of the first combinatorial modular polyketide library in Streptomyces (see PCT publication No. WO 98/49315, incorporated herein by reference).
The ability to control aspects of polyketide biosynthesis, such as monomer selection and degree of xcex2-carbon processing, by genetic manipulation of PKSs has stimulated great interest in the combinatorial engineering of novel antibiotics (see Hutchinson, 1998, Curr. Opin. Microbiol. 1: 319-329; Carreras and Santi, 1998, Curr. Opin. Biotech. 9: 403411; and U.S. Pat. Nos. 5,712,146 and 5,672,491, each of which is incorporated herein by reference). This interest has resulted in the cloning, analysis, and manipulation by recombinant DNA technology of genes that encode PKS enzymes. The resulting technology allows one to manipulate a known PKS gene cluster either to produce the polyketide synthesized by that PKS at higher levels than occur in nature or in hosts that otherwise do not produce the polyketide. The technology also allows one to produce molecules that are structurally related to, but distinct from, the polyketides produced from known PKS gene clusters.
Rapamycin is a macrocyclic polyketide that is produced by Streptomyces hygroscopicus (ATCC 29253, NRRL 5491) and Actinoplanes sp. N902-109 (see Drugs and the Pharmaceutical Sciences, Vol. 82, Biotechnology of Antibiotics, 2d Ed., ed. W. R. Strohl, Chapter 17, incorporated herein by reference). Rapamycin has antifungal, antitumor, and potent immunosuppressant activities, and is of significant interest for the treatment of autoimmune disease and prevention of rejection of organ and skin allografts (see Schwecke et al., August 1995, Proc. Natl. Acad. Sci. USA 92: 7839-7845, and references cited therein, incorporated herein by reference). The immunosuppressant activity arises from the ability of rapamycin to prevent the proliferative response of T cells to interleukin 2 bound at the interleukin 2 receptor (see Schwecke et al., supra). Hence, rapamycin offers an exciting opportunity to develop new classes of antifungal, antitumor, and immunosuppressant drugs
The number and diversity of rapamycin derivatives have been-limited due to the limited number of chemical modifications that can be performed on the complex molecule and the unavailability of recombinant host cells that produce rapamycin analogs. Genetic systems that result from engineering of the rapamycin biosynthetic genes would be valuable for creating novel compounds for pharmaceutical, agricultural, and veterinary applications. The production of such compounds could be more readily accomplished if recombinant host cells producing diverse rapamycin analogs were available. The present invention meets these and other needs.
The present invention provides recombinant methods and materials for expressing PKS enzymes and polyketide modification enzymes derived in whole and in part from the rapamycin biosynthetic genes in recombinant host cells. The invention also provides the polyketides produced by such PKS enzymes. Thus, in one embodiment, the invention is directed to recombinant materials comprising nucleic acids with nucleotide sequences encoding the rapamycin biosynthetic genes in which at least one domain, module, or protein encoded by a rapamycin PKS gene has been deleted, rendered inactive by mutation, or replaced by a different domain, module, or protein coding sequence.
In one embodiment, the invention provides recombinant host cells that produce a rapamycin derivative or analogue. In one embodiment, the rapamycin analogue is produced by a recombinant host cell that expresses a hybrid PKS comprising all or part of the rapamycin PKS and at least a part of a second PKS.
In a preferred embodiment, the host cell is Streptomyces hygroscopicus. 
The invention also provides novel polyketides, antitumor agents, antifungal agents, immunosuppressants and other useful compounds derived therefrom. The compounds of the invention can also be used in the manufacture of another compound. In a preferred embodiment, the compounds of the invention are formulated in a mixture or solution for administration to an animal or human.
These and other embodiments of the invention are described in more detail in the following description, the examples, and claims set forth below.